Insights into SCOC–FEZ1 complex formation

3.2.1.1 Approaches to find a minimal FEZ1 binding domain

S58 S301 S316

coiled coil C133

Fez1

1 392

Figure 3.34: FEZ1 domain

Human FEZ1 (392 residues) has properties of a natively unfolded protein.

It contains three glutamate rich regions and a conserved coiled coil domain in the C-terminal half of the protein [45, 110], see Figure 3.34. Coiled coils of both proteins are involved in the interaction of SCOC and FEZ1 [35].

As a protein with large intrinsically disordered parts, FEZ1 is a difficult target for crystallography. At first, I aimed to purify a complex of full-length FEZ1 with SCOC. However, coexpression inE.coli BL21 led to insoluble pro-teins, probably due to formation of inclusion bodies. I therefore prepared several FEZ1 constructs comprising parts of the coiled coil domain (see Ta-ble 3.10). According to COILS predictions, the ccd of FEZ1 starts around residue 230 and ends at residues 290–300. Noticable, this part of FEZ1 con-tains a lot of negatively charged residues and SCOC ccd also has a negative net charge. Hence, I cloned FEZ1 fragments with HisTag to add positive charge, so that SCOC and FEZ1 ccd would not repell each other in solution. FEZ1 ccd constructs were cloned with NdeI and XhoI restriction sites into vector pET22b (Novagen) using full length FEZ1 as template for PCR.

I expressed Strep-tagged SCOC (78–159) in combination with each of the FEZ1 constructs in E.coli BL21. Test expression was performed with Strep-tactin beads. The expression experiments revealed, that all of the His-tagged FEZ1 fragments were enriched together with Strep-tagged SCOC (78–159) in varying amounts. The minimal FEZ1 region interacting with SCOC (781–59) comprises residues 227–290 (Figure 3.35 A). Coexpression of FEZ1 (226–290) with SCOC (78–159) yielded a complex with equivalent amounts of both

pro-100 SCOC and its interaction partners

Table 3.10: FEZ1 fragments comprising the C-terminal ccd terminal

N-aa

terminal C-aa

length net

charge net charge HisTagwith

pI with His Tag

M227 L290 64 −5 −4/+2 5.94

M227 L295 69 −2 −1 6.65

H226 L290 65 −5/−4 −4/−3 6.03 H226 L295 70 −2/−1 −1/0 6.71 R225 L290 66 −4/−3 −3/−2 6.22

R225 L295 71 −1/0 0/+1 7.12

SCOC (78–159)

98 −5 4.98

A B

40 25 15 10 4.6

Figure 3.35: Test coexpression of Strep-SCOC (78–159) with His-FEZ1 ccd constructs

(A) Coexpression of Strep-SCOC (78–159) and His-FEZ1 (227–290) in LB medium at 37 °C (B) Coexpression of Strep-COC (78–159) and His-FEZ1 (226–290) in LB medium at 37 °C

3.2 Interaction of SCOC’s coiled coil domain with Arl1 and FEZ1 101 teins (Figure 3.35 B). Therefore, this complex was chosen for further investi-gation of the interaction.

3.2.1.2 Coexpression and copurification of SCOC–FEZ1 ccd complexes

5 6 7 8 9 10 11

Figure 3.36: Copurification of wt Strep-SCOC (78–159) with His-FEZ1 (226–290)

(A) Schägger gel of affinity chromatography with StrepTrap column (B) Schägger gel of size exclusion chromatography

Both FEZ1 (226–290) pET-22b and SCOC (78–159) pET-28a were co-trans-formed into BL21 by electroporation. The complex was expressed in LB medium at 37 °C. Cells were harvested and lysed. Affinity purification was conducted via StrepTrap chromatography. The protein complex eluted from the column in the same fractions 5–11 as SCOC (78–159) alone as shown in Figure 3.3 and Figure 3.36 B (see Section A.3.1 for elution profiles). The FEZ1 SCOC complex was further purified via size exclusion for SEC-MALLS (see Figure 3.36 A, C). The complex eluted from Superdex 75 16/60 column in a single peak. For further analysis, the complex was concentrated and used without freezing or further incubation, as it showed a tendency to precipitate and aggregate when protein samples were stored at 4 °C or −80 °C.

I assumed that oligomerization state and basic residues of SCOC would influence the interaction with FEZ1. Hence, SCOC double core mutants E93V/K97L and N125L/N132V, and arginine mutants R99E and R117E were also coexpressed and affinity purified with FEZ1 (226–290) via the same pro-tocol as above (see Section A.3.2 for gels of affinity purification). Figure 3.37

102 SCOC and its interaction partners

kDa 40 25 15 10 4.6

Figure 3.37: Coexpression of Strep-SCOC (78–159) constructs with His-FEZ1 (227–290)

Schägger gel with samples eluting from a StrepTrap column

shows copurification samples of all SCOC mutants with FEZ1 ccd. The tetra-meric N125L/N132V and tritetra-meric E93V/K97L mutants did not bind FEZ1 ccd showing that SCOC dimerization is crucial for SCOC–FEZ1 ccd complex formation. R117 is required for FEZ1 ccd interaction, since binding of R117E to FEZ1 ccd was almost completely abolished. The second arginine R99 is not important for complex formation, as R99E still interacted with FEZ1 ccd.

3.2.1.3 Characterization of SCOC–FEZ1 complexes

The formation of SCOC–FEZ1 ccd complex was analyzed by analytical gel filtration (Figure 3.38). The elution profile of the complex shows a considerable shift to shorter retention time compared to SCOC ccd alone. This indicates the formation of a stable complex with much larger molecular weight. Comparison to retention time of FEZ1 (227–190) was not possible, as the fragment was not expressed as soluble protein from E.coli, and could therefore not be purified.

The molecular weight of wt SCOC–FEZ1 ccd complex was analyzed with SEC-MALLS. The measurements yielded a molecular weight of 120.2±4 kDa for the SCOC–FEZ1 ccd complex and showed that the complex is homogeneous (Figure 3.39). FEZ1 is a dimer in solution [44, 45, 111]. Assuming that both proteins are dimers and that they interact with a 1:1 stoichiometry, there would be six copies of each protein in the SCOC–FEZ1 complex.

3.2 Interaction of SCOC’s coiled coil domain with Arl1 and FEZ1 103

-5,00 15,00 35,00 55,00 75,00 95,00 115,00

7,00 10,00 13,00 16,00 19,00 22,00

UV absorption (mAU)

Volume (mL)

FEZ1 ccd-SCOC ccd

SCOC

FEZ1 ccd-SCOC ccd SCOC

Figure 3.38: Analytical gel filtration of SCOC (78–159)—FEZ1 (227–290) complex

Elution profiles of SCOC (78–159)—FEZ1 (227–290 complex and SCOC (78–159) and the respective SDS-PAGE gels of the elution fractions

10000 100000 1000000 10000000

26 28 30 32

MW (g/mol)

time (min)

Figure 3.39: SEC-MALLS measurements of wt SCOC (78–

159)—FEZ1 (227–290) complex

Elution profiles and molecular weights from three measurements are shown for the complex.

104 SCOC and its interaction partners